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Abstract

Emerging evidence indicates that the kynurenine pathway (KP) contributes to neurodegenerative diseases associated with glial activation. Interferon (IFN)-γ is a potent activator of indole-2,3-dioxygenase, the first and rate-limiting enzyme of the KP. Our previous studies have shown that adult human astrocytes become neurotoxic when activated by IFN-γ. We now used high performance liquid chromatographymass spectrometry in both the positive- and negative- ionization mode of electrospray interface, to examine whether the IFN-γ-activated adult human astrocytes secrete neurotoxic KP metabolites, such as quinolinic acid (QUIN), 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HAA). Kynurenine was detected in cell culture supernatants of IFN-γ-stimulated astrocytes, but not in supernatants of unstimulated astrocytes. On the other hand, QUIN, 3-HK and 3-HAA were not detected in samples from either IFN-γ- stimulated or unstimulated cells. These results indicate that the KP may not be involved in the IFN-γ-induced neurotoxicity of adult human astrocytes. Therefore, neurotoxins other than KP metabolites could be responsible for the IFN-γ-induced astrocyte neurotoxicity.

Introduction

The kynurenine pathway KP (Figure 1)
metabolizes more than 95% of ingested
tryptophan, an essential amino acid, and
generates metabolites collectively called
kynurenines [1]. Several metabolites of the KP,
including kynurenine, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA)
and quinolinic acid (QUIN), are considered
to be neurotoxic and have essential roles in
activation of N-methyl-D-aspartate (NMDA)
receptor and in free radical production [2]. Since
NMDA receptor-mediated excitotoxicity and
excessive free radical production play important
roles in the mechanism of neuronal damage in
neurodegenerative disorders, the KP may be
a key player in their pathogeneses [3]. Indeed,
increasing evidence shows elevated levels of
QUIN, an endogenous NMDA agonist, in the
brain or cerebrospinal fluid of patients with
neurodegenerative disorders, such as Alzheimer’s
disease (AD) [4], Huntington’s disease [5] and
amyotrophic lateral sclerosis [6]. Both 3-HK and
3-HAA are endogenous generators of free radicals
[2,7]. These metabolites have been demonstrated
to reduce the viability of cultured cerebellar
granule neurons [8]. Moreover, increased brain
levels of 3-HK were demonstrated in Parkinson’s
disease patients [9] and elevated serum levels of
3-HK were reported in AD patients [10].

Figure 1: A schematic diagram of the kynurenine pathway of tryptophan metabolism in
human.

We have previously demonstrated that adult
human astrocytes become neurotoxic when
activated by interferon (IFN)-γ [11,12]. It is
well known that astrocytes are activated in neuroinflammatory processes associated with a
broad spectrum of neurodegenerative diseases
[13,14]. Interestingly, IFN-γ is one of the inducers
of indole-2,3-dioxygenase (IDO), which is the
first rate-limiting enzyme in the KP. In addition,
IFN-γ-stimulated fetal human astrocytes have
been shown to express IDO [15]. These findings
prompted us to examine whether the IFN-γ-
induced neurotoxicity of adult human astrocytes
involves neurotoxic metabolites of the KP. To
facilitate the detection of such metabolites in
the cell culture supernatants from IFN-γ-treated
astrocytes of adult human, we employed high
performance liquid chromatography (HPLC)-
mass spectrometry (MS) in both the positiveand
negative-ionization mode of electrospray
interface (ESI).

Materials and Methods

▪ Chemicals

Kynurenine, 3-HAA and QUIN were purchased
from Tokyo Chemical Industry (Tokyo, Japan),
while 3-HK was purchased from Sigma-Aldrich
(St. Louis, MO, USA). A mixture of the four
chemical standards was dissolved in 50% (w/v)
methanol and the final concentration of each
chemical was 50μg/mL. Four standard mixtures
were used for HPLC-MS analysis in both the
positive- and negative-ionization mode of ESI.
All the solvents used in this study were purchased
from Wako (Osaka, Japan). The organic solvents
(acetonitrile, ultrapure water, and formic acid)
for HPLC-MS were of liquid chromatography-
MS grade.

▪ Preparation of cell culture supernatant
samples

Adult human astrocytes were obtained from
three epileptic patients undergoing temporal
lobe surgery. The specimens were from normal
tissue overlying the epileptic foci. The use of
human brain materials was approved by the
Clinical Research Ethics Board for Human
Subjects of the University of British Columbia,
Canada. The epileptic patients were a 46 yearold
male, a 57 year-old female and a 26 yearold
male. Astrocytes were isolated as described
previously [11,12]. Primary astrocytes isolated
from each patient were considered as a separate
sample. Astrocytes were grown to confluence in
Dulbecco’s modified Eagle medium-nutrient
mixture F12 Ham (DMEM-F12) supplemented
with 10% fetal bovine serum (FBS), penicillin
(200 U/ml) and streptomycin (200 μg/ml) (all from ThermoFisher Scientific, Waltham, MA,
USA) on 10cm dishes in a cell incubator (37°C,
humidified 5% CO2). Astrocytes were rinsed
thrice with warmed FBS-free/phenol red-free
medium (ThermoFisher Scientific) and were
then cultured in 7 mL of the FBS- and phenol
red-free medium containing B27 supplement
(ThermoFisher Scientific). Subsequently, the cells
were treated with 50 U/ml of IFN-γ (PeproTech,
Rocky Hill, NJ, USA) or left unstimulated in
humidified 5% CO2, 95% air atmosphere at
37°C for 48 h. The cell-free supernatants were
collected and frozen at -80°C until analyzed
by HPLC-MS as described below. Under our
experimental conditions, immunocytochemistry
using antibody against the astrocytic marker glial
fibrillar acidic protein (GFAP) confirmed that
more than 99% cells were GFAP positive [12].

▪Metabolite extraction from the frozen
cell-free supernatants

The frozen cell-free supernatants were thawed
on an ice bath. Within 30 min of thawing the
supernatants, iced absolute methanol (0.3 mL)
was added to 0.1 mL of the sample. Subsequently,
the sample tube was vortexed and mixed to
remove the protein fraction. After centrifugation
of the mixture at 15,000 × g for 10 min at 4°C,
the supernatant samples were filtrated with
polytetrafluoroethylene hydrophilic membranes
(Merck Millipore, Japan).

▪ HPLC-MS analytical conditions

HPLC-MS was performed with an Agilent
1200 system (Agilent Technologies, Palo Alto,
CA) coupled to a Finnigan LTQ Orbitrap
XL (ThermoFisher Scientific), which was
equipped with an electrospray source operating
in the positive- and negative-ionization mode,
and with a lock-spray interface for accurate
mass measurement. Five different chemicals
(lidcaine, prochloraz, reserpine, bombesin, and
aureobasidin A) were employed as the lockmass
compounds for the positive-ionization
mode of ESI. A different set of five chemicals
(2,4 dichlorophenoxyacetic acid, ampicillin,
3-[(3-cholamidopropyl)dimethylammonio]
propanesulfonate, tetra-N-acetylchitotetraose,
and aureobasidin A) was employed as the lockmass
compounds for the negative-ionization
mode of ESI. An aliquot of the extracted sample
(5 μL) was injected into a TSKgel column ODS-
100V (5 μm, 3 x 50 mm; Tosoh, Tokyo, Japan)
with mobile phases which consisted of 0.1%
(v/v) aqueous formic acid (solvent A) and 0.1%
(v/v) formic acid in acetonitrile (solvent B). The gradient program was as follows: 3-97% solvent
B for the first 15 min, 97% solvent B for the next
5min, and 3% solvent B from 20 to 25 min, with
a flow rate of 0.4 mL/min. The column oven
temperature was set at 40°C. HPLC-MS analysis
was performed in both the positive-ionization
mode and negative-ionization mode of ESI.

▪ Data analysis of HPLC-MS

All data obtained from the HPLC-MS analysis
were acquired with the Xcalibur™ software
(ThermoFisher Scientific). To determine
the lowest concentrations detectable in our
system for the four KP metabolites, namely,
kynurenine, 3-HK, 3-HAA and QUIN in cell
culture supernatants, we focused the HPLCMS
analysis on the adduct ion forms ([M+H]+ or [M+H]-) which occur in each of the four
targeted KP metabolites using the Xcalibur™
software. We calculated the peak area values for
the four KP metabolites based on extracted ion
chromatogram of m/z [M+H] ± 3 ppm.

▪ Determination of the lowest detectable
concentrations for the KP metabolites
using the HPLC-MS analysis

To determine the lowest detectable
concentrations for the four KP metabolites
(kynurenine, 3-HK, 3-HAA and QUIN), we
searched for the detectable concentrations of
authentic compounds by testing several different
concentrations (ranging from 10 pM to 10 μM)
of analytical-grade standards in our HPLC-MS
analytical system. We used cell culture medium
to prepare solutions of kynurenine, 3-HK,
3-HAA and QUIN at the final concentrations of
10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 100 pM
and 10 pM. Each of the above standard dilutions
of the four KP metabolites (kynurenine, 3-HK,
3-HAA and QUIN) was independently injected
three times into the HPLC-MS equipment in
5-μL aliquots. After HPLC-MS analysis, the
peak area of m/z [M+H] ± 3 ppm was divided
by the area of authentic compounds, to calculate
the peak area values for these compounds. In
the ESI positive-ionization mode of our HPLCMS
analytical system, the peak area values for
kynurenine, 3-HK, 3-HAA and QUIN were
determined to be m/z=209.0921 ± 3 ppm, m/
z=225.0870 ± 3 ppm, m/z=154.0499 ± 3 ppm,
and m/z=168.0291 ± 3 ppm, respectively. In the
ESI negative-ionization mode of our HPLCMS
analytical system, the peak area values for
kynurenine, 3-HK, 3-HAA and QUIN were
determined to be m/z= 207.0775± 3 ppm,
m/z=223.0724 ± 3 ppm, m/z= 152.0353± 3 ppm, and m/z=166.0146± 3 ppm, respectively. Table 1 and Supplementary Table 1 show the
lowest detectable concentrations for the four
KP metabolites in our HPLC-MS analytical
system in the positive-ionization and negativeionization
modes of ESI.

KP metabolizes (abb. name)

Chemical composition fourmula

Detected full mass (m/z)

Concentration

Elution time

Peak Area

Sample (n1)

Sample (n2)

Sample (n3)

kynurenine

C10H12N2O3

209.0923
[M+H]+

10 µM

2.63 min

19558367

20210107

19162270

1 µM

2017830

1970963

2018336

100 nM

199352

196436

192607

10 nM

N.D.

N.D.

N.D.

1 nM

N.D.

N.D.

N.D.

100 pM

N.D.

N.D.

N.D.

10 pM

N.D.

N.D.

N.D.

control

N.D.

N.D.

N.D.

3-hydroxykynurenine
(3-HK)

C10H12N2O4

225.0872
[M+H]+

10 µM

1.53 min

6597988

7080201

6555178

1 µM

532920

561289

492719

100 nM

N.D.

N.D.

N.D.

10 nM

N.D.

N.D.

N.D.

1 nM

N.D.

N.D.

N.D.

100 pM

N.D.

N.D.

N.D.

10 pM

N.D.

N.D.

N.D.

control

N.D.

N.D.

N.D.

3-hydroxyanthranilic acid
(3-HAA)

C7H7NO3

154.0501
[M+H]+

10 µM

4.17 min

12345992

12211420

12658353

1 µM

1157383

1171459

1319920

100 nM

N.D.

195202

216426

10 nM

N.D.

N.D.

N.D.

1 nM

N.D.

N.D.

N.D.

100 pM

N.D.

N.D.

N.D.

10 pM

N.D.

N.D.

N.D.

control

N.D.

N.D.

N.D.

quinolinic acid
(QUIN)

C7H5NO4

168.0293
[M+H]+

10 µM

1.32 min

156334

169688

105259

1 µM

N.D.

N.D.

N.D.

100 nM

N.D.

N.D.

N.D.

10 nM

N.D.

N.D.

N.D.

1 nM

N.D.

N.D.

N.D.

100 pM

N.D.

N.D.

N.D.

10 pM

N.D.

N.D.

N.D.

blank control

N.D.

N.D.

N.D.

N.D. describes as not detected for the tageted metabolite
Control describes as using only not detected for the tageted metabolite.

To determine the lowest detectable concentrations for the four KP metabolites, kynurenine, 3-HK, 3-HAA and QUIN, several different concentrations
(ranging from 10 pM to 10 μM in cell culture medium) of analytical-grade standards were tested in our HPLC-MS analytical system.
All HPLC-MS data were acquired by using the positive-ionization mode of ESI.

Table 1: Determination of the lowest detectable concentrations for the four KP metabolites using the HPLC-MS analysis
with the positive ionization mode of ESI.

Results

We first, by employing authentic compounds,
confirmed the detection of the four KP
metabolites, kynurenine, 3-HK, 3-HAA and
QUIN, in our HPLC-MS analytical system
with the positive-ionization mode (Figure 2) and negative-ionization mode (Supplementary
Figure 1) of ESI. Next, cell culture supernatants
of adult human astrocytes were analyzed by
using our HPLC-MS system in both the
positive-ionization mode (Figure 3) and
negative-ionization mode (Supplementary
Figure 2) of ESI. Kynurenine was detected in cell
culture supernatants of adult human astrocytes
stimulated with IFN-γ, but not in supernatants
of unstimulated astrocytes. QUIN, 3-HK and
3-HAA were not detected in either IFN-γ-
stimulated samples or in unstimulated control
samples (Figure 3 and Supplementary Figure 2).

The HPLC-MS analysis system used in the present study differs from those used in previous
investigations which detected QUIN, 3-HK
and 3-HAA in cell culture supernatants of IFN-
γ-treated human monocytes [16] and QUIN
in rat plasma [17]. Therefore, we prepared the
KP metabolite solutions dissolved in cell culture
medium at several different concentrations
(ranging from 10 pM to 10 μM), in order to
determine the lowest detectable concentrations
for the four KP metabolites in our HPLC-MS
system. The lowest detectable concentrations
for kynurenine, 3-HK, 3-HAA and QUIN were
determined to be 100 nM, 1 μM, 100 nM,
and 10 μM, respectively, under the positiveionization mode of ESI (Table 1). Under the
negative-ionization mode of ESI, the lowest
detectable concentration for all four metabolites
was determined to be 1 μM (Supplementary
Table 1).

Discussion

Recent evidence indicates that the KP
contributes to the pathogenesis in
neurodegenerative diseases associated with
glial activation. It has also been reported
that IFN-γ activates IDO, the initial ratelimiting
enzyme of the KP (reviewed in Tan, et al. [18] ). Our previous studies have
shown that IFN-γ-activated adult human
astrocytes secrete neurotoxin(s) [11,12]. Now
we aimed to determine whether the KP is
involved in the IFN-γ-induced neurotoxicity
of adult human astrocytes. Three of the KP
metabolites, QUIN, 3-HK and 3-HAA have been detected in cell culture supernatants of
human monocytes treated with IFN-γ by using
an HPLC-ESI-positive-MS method [16].
Another study demonstrated that the negative
ionization approach provided the highest
sensitivity for the detection of QUIN in rat
plasma [17,18]. We therefore performed the HPLC-MS in both the positive- and negativeionization
mode of ESI. Nevertheless, we were
unable to detect the neurotoxic kynurenines
QUIN, 3-HK or 3-HAA in supernatants from
IFN-γ-stimulated adult human astrocytes by
using either the positive or negative ESI mode.
These results indicate that the KP may not be
involved in the IFN-γ-induced neurotoxicity
of adult human astrocytes, even though we
cannot completely rule out the possibility that
very low concentrations of the KP metabolites
at undetectable levels in our HPLC-MS
system contribute to the neurotoxic activity
of astrocytes. Our results also suggest that
increased levels of the neurotoxic kynurenines,
including QUIN, shown in neurodegenerative
disorders may stem from activated microglia,
not astrocytes, since cultured human
microglia have been demonstrated to
produce QUIN [19]. Although the exact
roles of the KP in astrocytes remain unclear,
a study using pharmacological inhibition
of the KP suggests that the KP directly
facilitates the maintenance of intracellular
levels of nicotinamide adenine dinucleotide
and sirtuin deacetylase-1 activity in human
astrocytes [20].

Very few studies have examined astrocytic
production of the neurotoxic KP metabolites
using human cells. Our results showing
undetectable levels of QUIN, 3-HK and 3-HAA
in supernatants from IFN-γ-stimulated astrocytes
of adult human are consistent with the previous
limited studies. Guillemin, et al. demonstrated
that fetal human astrocytes did not express the
mRNA encoding kynurenine hydroxylase even
when these cells were stimulated with IFN-γ
[15]. Since kynurenine hydroxylase converts
kynurenine into 3-HK, absence of this enzyme
renders fetal human astrocytes unable to produce
the downstream KP metabolites, including
3-HK, 3-HAA and QUIN (Figure 1). Indeed,
it has been shown that cultured fetal human
astrocytes do not synthesize detectable levels of
QUIN or 3-HK [15]. Although Guillemin and
colleagues did not examine whether 3-HAA
is secreted by IFN-γ-stimulated fetal human
astrocytes, we confirmed no detectable levels of
3-HAA in supernatants from IFN-γ-stimulated
adult human astrocytes. Heyes, et al. also
reported that human astrocytoma U373-MG
cells activated by IFN-γ showed no detectable
production of QUIN [21]. Based on these
findings, it can be concluded that, regardless of their age or proliferative status, human
astrocytes do not produce high levels of 3-HK,
3-HAA or QUIN even when stimulated with
IFN-γ. In contrast to their human counterparts,
gerbil astrocytes have been shown to produce
increased levels of QUIN when stimulated
with lipopolysaccharide [22]. Therefore,
astrocytic capacity to produce neurotoxic
kynurenines appears to vary between species
and between stimulants.

It still needs to be established conclusively
which neurotoxins secreted in supernatants
are responsible for the IFN-γ-induced
neurotoxicity of adult human astrocytes. This
study was performed to discover whether
toxic metabolites of the KP are responsible
for the human astrocyte neurotoxicity.
However, our results do not support the
hypothesis that the KP mediates the IFN-
γ-induced neurotoxicity of adult human
astrocytes. Therefore, neurotoxins other than
KP metabolites should be responsible for the
IFN-γ-induced astrocyte neurotoxicity. It is
likely that no single molecule is responsible
for the astrocytic neurotoxicity, since
microarray analysis revealed 1192 genes
that were differentially expressed in murine
astrocytes in response to IFN-γ by a factor
of 1.5 or greater. Moreover, approximately
one-fourth of these genes were of unknown
identity [23]. Further exploration of the
compounds responsible for the astrocyte
neurotoxicity is clearly warranted since an
understanding of these mechanisms could
lead to the discovery of new therapeutic
targets for neurodegenerative diseases.

Author Contributions

SH, HS and AK participated in the design
of the study. SH, DN and AK carried out all
experiments, collected the data and performed
the statistical analysis. SH, HS and DN
interpreted the data. SH, HS and AK wrote the
manuscript. TM, RW, MH, JH and AK revised
the manuscript. All authors read and approved
the final version of manuscript.

Acknowledgements

Sincere appreciation is extended to Drs. Edith
and Patrick McGeer for their invaluable support.
This research was supported by Grant-in-Aid for
Scientific Research #24591721 (SH).